The focus of our research is to determine how signal transduction regulates directed motility and behavior in the bacterium Myxococcus xanthus using an integrated approach that combines biochemistry, genetics, and cell biology. M. xanthus is an excellent model system to address fundamental questions concerning cell-cell signaling and directed movement as cells form multicellular biofilms and fruiting bodies as part of a complex life cycle. M. xanthus fruiting bodies are similar in many ways to biofilms formed by some pathogenic bacteria and are of public health interest since biofilms render bacteria resistant to antibiotics and are very difficult to treat in patients. Biofilm and fruiting body formation require the activity of chemosensory systems to direct cell movements. Previously, we have shown that the Frz chemosensory pathway regulates both vegetative swarming and developmental aggregation by controlling the reversal frequency of cells. Cell reversals in Myxococcus, like tumbling in flagellated bacteria, allows cells to reorient themselves and to bias directional motility based on the temporal sensing of stimuli. In the last grant period, our studie of the Frz system have allowed us to identify important proteins responsible for the functioning of the gliding motility engines. Our first specific aim is to continue our characterization of the dynamics of the motor protein AglR, a MotA homolog, at single molecule resolution. For these experiments, we are tracking the AglR motility complexes with photoactivatable localization microscopy (PALM) at millisecond intervals. These experiments should provide important information on this novel motility system. The techniques being utilized are innovative because they permit analysis of the dynamics of single molecule motors at nanometer resolution. Our second specific aim is to finalize our study of the Frz pathway by identifying the output of the pathway: the proteins that interact directly with the phosphorylated receiver domain, FrzZ. The output of the pathway is responsible for transmitting information from the Frz chemosensory pathway to the motors and the controllers of cell polarity, reversing the gliding motility and Type IV pili engines of M. xanthus. These experiments are significant because the coupling between the chemosensory system and the gliding motility engines is very different from the E. coli paradigm and likely to be utilized by other bacterial systems.